Indiana University-Purdue University Indianapolis (IUPUI) / Pancreatic insulin-secreting beta cell failure is central to the development of diabetes. Therapeutic applications targeted at understanding and manipulating beta cell destruction mechanisms should enhance the preservation of functional beta cell mass and prevent diabetes. To this end, we have demonstrated that diabetogenic assaults (e.g., endoplasmic reticulum stress, glucolipotoxicity, and pro-inflammatory cytokines) attenuate the activation of beta cell pro-survival signaling pathways via a stress-inducible molecule called Mitogen-inducible gene 6 (Mig6). We discovered that the overabundance of Mig6 exacerbates stress-induced beta cell apoptosis and inhibits insulin secretion. Conversely, the deficiency of Mig6 partially protected beta cells from DNA damage-induced cell death. Further, we established that Mig6 haploinsufficient mice retained islet integrity and function and exhibited greater beta cell mass recovery following treatment with multiple low doses of the beta cell toxin streptozotocin. These data suggest that Mig6 may be a therapeutic target for beta cell preservation in diabetes.

Indiana University-Purdue University Indianapolis (IUPUI) / Pancreatic ductal adenocarcinoma (PDA) is a deadly disease due to early metastasis and resistance to chemotherapy. PDA is commonly associated with a dense desmoplastic stroma, which forms a protective niche for cancer cells. Tissue transglutaminase (TG2), a Ca2+-dependent enzyme, is abundantly expressed in pancreatic cancer cells and crosslinks proteins through acyl-transfer transamidation between glutamine and lysine residues. The objective of the study was to determine the functions of TG2 in the pancreatic stroma. Orthotopic pancreatic xenografts and co-culture systems tested the mechanisms by which the enzyme modulates tumor-stroma interactions. We showed that TG2 secreted by cancer cells is enzymatically active and renders the stroma denser by crosslinking collagen, which in turn activates fibroblasts and stimulates their proliferation. Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) are transcription factors involved in mechanotransduction. The TG2-mediated fibrosis-rich, stiff microenvironment conveys mechanical cues to cancer cells leading to activation of YAP and TAZ, promoting cell proliferation and tumor growth. Stable knockdown of TG2 in pancreatic cancer cells led to decreased size of pancreatic xenografts and increased sensitivity of xenografts to gemcitabine. Taken together, our results demonstrate that TG2 secreted in the tumor microenvironment orchestrates the crosstalk between cancer cells and the stroma, fundamentally impacting tumor growth and response to chemotherapy. Our study supports TG2 inhibition in the pancreatic stroma as a novel strategy to block pancreatic cancer progression.

The authors report a case of endometriosis that presented as a cystic mass in the tail of the pancreas, leading to extensive evaluation and ultimately a major surgical resection. The diagnosis was made by histopathological evaluation, revealing endometrial glands and stroma in the wall of the mass with hemorrhagic fluid in the cystic lumen, compatible with pancreatic involvement by an endometrial cyst.

No description available.

Intracellular hyperthermic therapy may prove to be a unique and novel approach to the management of pancreatic cancer. Utilizing the principle of photothermal destruction, selective killing of cancer cells with minimal injury to normal tissues may be possible. This dissertation investigated the role of antibody targeted metal nanoparticles and the cytotoxic effects of nonionizing radiofrequency fields in pancreatic cancer. Cancer cell death was induced by heat release from intracellular metal nanoparticles after radiofrequency field exposure. Fluorescent and gold nanoparticles were delivered with two antibodies, cetuximab and PAM-4, to pancreatic cancer cells in vitro and mouse xenografts in vivo. Selective delivery of these nanoparticles induced cell death in vitro and decreased tumor burden in vivo after whole animal RF field exposure. This occurred through both apoptosis and necrosis. In addition, activated caspase-3 was increased after antibody treatment and RF field exposure. Furthermore, although there was non-specific uptake by the liver and spleen in vivo, there was no evidence of acute or chronic toxicity in the animals. These results are in agreement with the principle that malignant cells are more thermally sensitive than normal cells or tissues. Selective intracellular delivery of metal nanoparticles coupled with whole body RF field exposure may be a beneficial therapy against micrometastases and unresectable pancreatic cancer in the future. Further studies are planned with more specific antibodies, other nanoparticles, and other cancer targets.

Pancreatic adenocarcinoma (PDAC) putatively arises from the pancreatic duct, thus usage of the normal human pancreatic duct epithelial (HPDE) cell line is an ideal model to examine the successive accumulation of genetic alterations involved in carcinogenesis. KRAS mutations have been reported in 90% of PDACs. Oncogenic KRAS elicits activation of downstream pathways involved in survival, motility, and cell cycle progression. KRASG12V introduction in the HPDE cell line upregulates Lipocalin-2 (LCN2) expression. LCN2 has been identified in numerous carcinomas and is associated with survival, tumorigenicity, and invasion. In this work, LCN2 was found to be commonly expressed in high grade pancreatic duct neoplastic precursor lesions and PDAC illustrating its potential as a biomarker. Moreover, in vitro and in vivo studies demonstrate that high LCN2 expression promotes gemcitabine resistance, MMP-9 activity, angiogenesis, and tumorigenicity. Loss of Smad4 function is found in 55% of PDAC cases. Smad4 is a critical component in the TGF-β signaling which mediates the transcription of genes involved in processes such as cell cycle arrest, apoptosis, and invasion. This work examined the consequences of KRASG12V expression and Smad4 loss in the HPDE model. Cellular invasion was promoted by KRASG12V expression or knocking down Smad4 by 80% in the HPDE model. A TGF-β resistant HPDE cell line, TβR, was shown to lack Smad4 expression due to deletion, promoter methylation, and nonsense mutation. KRASG12V expression in the TβR model (TβR KRAS) promoted neoplastic transformation and tumour formation in immunodeficient mice with complete penetrance. Smad4 expression in the TβR KRAS cell line reinstated TGF-β signaling, delayed tumour formation, and decreased metastatic spread. This study provides evidence that Smad4 acts as a restriction point in the transformation of HPDE cells. Overall, this work examines the contribution of genes involved in transformation, and identifies a potential therapeutic and diagnostic biomarker in PDAC.

Embryonic stem (ES) cells were used as a model system to understand the signalling events in pancreas development. ES cells were differentiated through known precursor stages towards the tissue of interest in order to recapitulate development in vitro. Thus, protocols directing differentiation of mouse ES cells towards definitive endoderm and pancreatic β-cells were developed. A combination of activin A and bone morphogenic protein 4 resulted in a population of enriched cells expressing genetic markers of definitive endoderm. In vitro differentiation of ES cells into functional pancreatic β-cells has only been partially successful, as it results in cells that are not fully differentiated or functional. This might be due to a lack of cues emanating from surrounding cells present in the developing pancreas. Conditioned media from the mouse MIN6 β-cell line were used on the basis that differentiated β- cells might send out signals affecting the differentiation of the surrounding islet cells. Mouse ES cells were enriched in definitive endoderm and then treated with MIN6 conditioned medium. Gene expression of the β-cell markers Insulin1, Insulin2, and Glucose transporter 2 was significantly increased relative to the untreated control group after 10 days of treatment with conditioned medium. This result was specific for conditioned medium from MIN6 cells as conditioned medium from a kidney-, a neuronal-, and an exocrine pancreatic cell line had no effect. In order to characterise the secreted factor(s) the conditioned medium was subjected to protein precipitation. The pancreatic differentiation factor was present in a protein fraction, suggesting that the factor(s) was proteinaceous. The protein in question was neither proinsulin nor insulin. This knowledge will support the efficient generation of insulin-secreting cells for diabetes therapy.

Embryonic stem (ES) cells represent a possible source of islet tissue for the treatment of diabetes.  Achieving this goal will require a detailed understanding of how the transcription factor cascade initiated by the homeodomain transcription factor Pdx1 culminates in pancreatic beta-cell development.  Here we describe a genetic approach that enables fine control of Pdx1 transcriptional activity during endoderm differentiation of mouse ES cell.  By activating an exogenous Pdx1VP16 protein in populations of cells enriched in definitive endoderm we show a distinct lineage-dependent requirement for this transcription factor’s activity.  mimicking the natural biphasic pattern of Pdx1 expression was necessary to induce an endocrine pancreas-like cell phenotype, in which 30% of the cells were beta-cell-like.  Cell markers consistent with the different beta-cell differentiation stages appeared in a sequential order following the natural pattern of pancreatic development.  Furthermore, the differential beta-like cells secreted C-peptide (insulin) in response to KC1 and IBMX, suggesting that following a natural path of development in vitro represents the best approach to generate functional pancreatic cells.  Together these results reveal for the first time a significant effect of the timed expression of Pdx1 on the non-beta cells in the developing endocrine pancreas.  Collectively, we show that this method of <i>in vitro</i> differentiation provides a template for inducing and studying ES cell differentiation into insulin-secreting cells.

A Thesis submitted to The University of Arizona College of Medicine - Phoenix in partial fulfillment of the requirements for the Degree of Doctor of Medicine. / Metastatic, treatment‐resistant pancreatic ductal adenocarcinoma (PDAC) is a rapidly fatal disease that typically carries a bleak prognosis. Contrast‐enhanced CT is the current standard of care tool for imaging evaluation, and repeat imaging is routinely performed in clinical trials. The availability of these imaging data render them exploitable for further analysis. CT texural analysis (CTTA), a quantitative tool for examining a region of interest on CT and generating statistical parameters based on gray‐level pixel data, is powerful technique that has been studied in other cancers and shown to correlate with features such as tumor grade, stage, and prognosis. However, the application of CTTA to PDAC has not been studied. Given the paucity of diagnostic tests to guide therapy, validated CTTA biomarkers could be immensely useful. Identifying PDAC variants that have a relative deficit in DNA repair might allow these cancers to be treated with targeted cytotoxic regimens sooner. Additionally, identifying prognostic CTTA parameters would be useful in gauging the severity of disease. We sought to perform quantitative textural analysis on CT imaging from a clinical trial cohort of patients with metastatic, treatment‐resistant PDAC. We aimed to correlate CTTA features to molecular profiling results (copy number variations obtained by array CGH) and clinical features (overall survival). Metastatic tumor sites from patients with treatment‐resistant PDAC were biopsied and molecularly profiled. Intrachromosal copy number were assessed by CGH in tumor specimens, and patients were treated based on these individual molecular profiling results. Pre‐biopsy portal‐venous phase and non‐contrast CT scans were obtained for retrospective analysis (n=15). CTTA was performed by drawing regions of interest around the primary pancreas adenocarcinoma and the normal pancreas tissue. CTTA parameters including mean positive pixels, entropy, kurtosis, and skewness were derived using the TexRAD platform at texture filtering densities of 0, 2, 3, 4, 5, and 6 pixels. CTTA values were then compared to intrachromosomal copy number variation (CNV) per tumor and overall survival (OS) post treatment using a Spearman’s rank correlation coefficient. Additional linear regression analysis was performed for positive correlations, and a Kaplan‐Meier statistic was generated for OS using median CTTA entropy. Multivariate analyses for CNV and OS were also performed. CNV were negatively correlated with the kurtosis value of the primary tumor mass using medium texture filtering (p=0.034, n=15). Linear regression revealed a significant negative correlation between kurtosis and CNV (p=0.038). Secondary analysis of the normal pancreas using coarse texture filtering revealed that increasing entropy was associated with decreased OS (p=0.0014, n=12). Using median entropy as a cutoff value (median: 4.165), median OS was greater in the entropy < 4.165 group versus the entropy > 4.165 group (179 days v 43 days; 95% CI 73.137 – 166.87; p=0.004, n=12). This exploratory study with admittedly limited sample size raises interesting questions about the use of CTTA parameters as diagnostic tools and/or biopsy adjuncts in assessing PDAC susceptibility to commercially available cytotoxics. Secondarily, entropy, a potential marker of heterogeneity and inflammation in the normal pancreas, represents an intriguing possibility for gauging prognosis.

Intraductal papillary mucinous neoplasms (IPMN) are pancreatic lesions with uncertain biologic behavior. This study sought objective, accurate prediction tools, through the use of quantitative histopathological signatures of nuclear images, for classifying lesions as chronic pancreatitis (CP), IPMN, or pancreatic carcinoma (PC). Forty-four pancreatic resection patients were retrospectively identified for this study (12 CP; 16 IPMN; 16 PC). Regularized multinomial regression quantitatively classified each specimen as CP, IPMN, or PC in an automated, blinded fashion. Classification certainty was determined by subtracting the smallest classification probability from the largest probability (of the three groups). The certainty function varied from 1.0 (perfectly classified) to 0.0 (random). From each lesion, 180 +/- 22 nuclei were imaged. Overall classification accuracy was 89.6% with six unique nuclear features. No CP cases were misclassified, 1/16 IPMN cases were misclassified, and 4/16 PC cases were misclassified. Certainty function was 0.75 +/- 0.16 for correctly classified lesions and 0.47 +/- 0.10 for incorrectly classified lesions (P = 0.0005). Uncertainty was identified in four of the five misclassified lesions. Quantitative histopathology provides a robust, novel method to distinguish among CP, IPMN, and PC with a quantitative measure of uncertainty. This may be useful when there is uncertainty in diagnosis.